The orientation and dynamics of estradiol and estradiol oleate in lipid membranes and HDL disc models.

Estradiol (E2) and E2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E2 and E2 oleate in membranes and reconstituted HDLs, finding that E2 and E2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membrane's z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E2 oleate is almost exclusively oriented at an angle of ∼60° to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E2 and E2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E2 interactions determine the localization of the molecule. The structural mapping of E2 and E2 oleate may provide a design platform for specific E2-HDL-targeted pharmacological therapies.

Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes.

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations-as well as their extensive fluctuations-suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.

A reinterpretation of neutron scattering experiments on a lipidated Ras peptide using replica exchange molecular dynamics.

The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function.

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation.

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.

Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations.

Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.